Microwell design and fabrication for generation of cell culture aggregates

ABSTRACT

A cell culture apparatus may include a substrate defining a well. The well may define an interior surface, an exterior surface, an upper aperture and a nadir. The substrate may define a thickness between the interior and exterior surfaces that has a thickness proximate the nadir that is greater than or equal to a thickness proximate the upper aperture.

This is a continuation of International Application Serial No.PCT/US15/58123, filed on Oct. 29, 2015, which claims benefit of priorityto U.S. Provisional Application Ser. No. 62/072,019 filed on Oct. 29,2014 the contents of which are relied upon and incorporated herein byreference in their entirety.

FIELD

The present disclosure relates to apparatuses, systems and methods forculturing cells.

TECHNICAL BACKGROUND

Cell culture techniques that encourage formation of 3D aggregates orspheroids have been strongly advocated over traditional monolayerculture techniques due to the increased number of applications. However,some conventional cell culture apparatuses currently used in formingspheroids make imaging techniques difficult.

BRIEF SUMMARY

In accordance with various embodiments of the present disclosure,apparatuses having wells for use in culturing cells to promote theformation of spheroids are described herein. Embodiments of apparatusesdescribed herein have well geometries which minimize light distortionthat can occur in conventional apparatuses used for culturing spheroids,allowing for improved imaging quality of spheroids grown in the wells.

In various embodiments, the disclosure describes a cell cultureapparatus having a substrate defining a well. The well defines aninterior surface, an exterior surface, an upper aperture and a nadir.The substrate defines a thickness between the interior surface and theexterior surface. A thickness of the substrate proximate to the nadir isgreater than or equal to a thickness of the substrate proximate to theupper aperture.

In various embodiments, the disclosure describes a cell cultureapparatus including a reservoir comprising a bottom and an enclosingsidewall. The bottom is defined by a plurality of wells. Each well ofthe plurality of wells defines an interior surface, an exterior surface,an upper aperture and a nadir. The well defines a thickness between theinterior surface and the exterior surface. A thickness of the wellproximate to the nadir is greater than or equal to a thickness of thewell proximate to the upper aperture.

In various embodiments, the disclosure describes a cell cultureapparatus including a substrate defining a well. The well defines aninterior surface, an exterior surface, an upper aperture and a nadir.The substrate defines a thickness between the interior surface and theexterior surface. The thickness is configured to correct for refractionof light passing into the interior surface and out of the exteriorsurface when the well contains a water-based composition. Inembodiments, the water-based composition is a composition employed incell culture or cell assays. For example, a water-based composition caninclude a cell culture medium, buffers or other solutions or mixturesemployed in cell assays.

In various embodiments, the disclosure describes a cell cultureapparatus includes a substrate defining a well. The well defines aninterior surface, an exterior surface, an upper aperture and a nadir. Ashape of the exterior surface is configured to correct for refraction oflight passing into the interior surface and out of the exterior surface.

In some embodiments, provided herein is a cell culture apparatuscomprising: a substrate defining a well, wherein the well defines aninterior surface, an exterior surface, an upper aperture and a nadir,wherein the substrate defines a thickness between the interior surfaceand the exterior surface, wherein a thickness of the substrate proximateto the nadir is greater than or equal to a thickness of the substrateproximate to the upper aperture. In some embodiments, the thickness ofthe substrate proximate to the nadir is greater than the thickness ofthe substrate proximate to the upper aperture. In some embodiments, thethickness of the substrate increases continuously from proximate theupper aperture to the nadir. In some embodiments, the thickness of thesubstrate proximate to the nadir is equal to the thickness of thesubstrate proximate to the upper aperture. In some embodiments, thethickness of the substrate remains constant from proximate the upperaperture to the nadir.

In some embodiments, the well defines an axis between the nadir and acenter of the upper aperture, wherein the well is rotationallysymmetrical about the axis.

In some embodiments, the upper aperture defines a distance across theupper aperture, wherein the distance across the upper aperture is in arange from 100 micrometers to 3000 micrometers.

In some embodiments, the thickness of the substrate at any location fromproximate the upper aperture to the nadir is in a range from 10micrometers to 1000 micrometers.

In some embodiments, the interior surface is defined by a hemisphericalshape, wherein the hemispherical shape defines a radius in a range from50 micrometers to 1500 micrometers.

In some embodiments, the exterior surface is configured to transmitlight with a divergent angle smaller than the numerical aperture of theimaging system. For example for 4× Plan Achromat magnification objectivewith numerical aperture 0.1, light should pass substantially parallel(i.e., 5.7° or less) to a direction that the light was received by theinterior surface when the well contains a cell culture medium. Ingeneral, the maximum divergence angle of the light passing through thewell with cell culture should not exceed the acceptance cone of anobjective.

In some embodiments, the shape of the interior surface and the shape ofthe exterior surface are configured to minimize refraction of light thatpasses there between when the well contains a cell culture medium.

In some embodiments, the well is non-adherent to cells.

In some embodiments, the interior surface is configured such that cellscultured therein form a spheroid.

Also provided herein is a cell culture apparatus comprising: a reservoircomprising a bottom and an enclosing sidewall, wherein the bottom isdefined by a plurality of wells, wherein each well of the plurality ofwells defines an interior surface, an exterior surface, an upperaperture and a nadir, wherein the well defines a thickness between theinterior surface and the exterior surface, wherein a thickness of thewell proximate to the nadir is greater than or equal to a thickness ofthe well proximate to the upper aperture.

Further provided herein is a cell culture apparatus comprising: asubstrate defining a well, wherein the well defines an interior surface,an exterior surface, an upper aperture and a nadir, wherein thesubstrate defines a thickness between the interior surface and theexterior surface, wherein the thickness is configured to correct forrefraction of light passing into the interior surface and out of theexterior surface when the well contains a water-based composition.

Further provided herein is a cell culture apparatus comprising: asubstrate defining a well, wherein the well defines an interior surface,an exterior surface, an upper aperture and a nadir, wherein a shape ofthe exterior surface is configured to correct for refraction of lightpassing into the interior surface and out of the exterior surface.

Further provided herein are uses of any of the above for the growthand/or imaging or assessment of cells (e.g., spheroids).

Additional features and advantages of the subject matter of the presentdisclosure will be set forth in the detailed description which follows,and in part will be readily apparent to those skilled in the art fromthat description or recognized by practicing the subject matter of thepresent disclosure as described herein, including the detaileddescription which follows, the claims, as well as the appended drawings.

It is to be understood that both the foregoing general description andthe following detailed description present embodiments of the subjectmatter of the present disclosure, and are intended to provide anoverview or framework for understanding the nature and character of thesubject matter of the present disclosure as it is claimed. Theaccompanying drawings are included to provide a further understanding ofthe subject matter of the present disclosure, and are incorporated intoand constitute a part of this specification. The drawings illustratevarious embodiments of the subject matter of the present disclosure andtogether with the description serve to explain the principles andoperations of the subject matter of the present disclosure.Additionally, the drawings and descriptions are meant to be merelyillustrative, and are not intended to limit the scope of the claims inany manner.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of specific embodiments of thepresent disclosure can be best understood when read in conjunction withthe following drawings, where like structure is indicated with likereference numerals and in which:

FIG. 1 is a schematic cross-sectional view of an embodiment of a cellculture apparatus including a plurality of wells.

FIG. 2A is a cross-sectional view of an embodiment of one well of a cellculture apparatus.

FIG. 2B is a cross-sectional view of an embodiment of one well of a cellculture apparatus.

FIG. 3A is a cross-sectional view of an embodiment of one well of a cellculture apparatus.

FIG. 3B is a cross-sectional view of an embodiment of one well of a cellculture apparatus.

FIGS. 4A-4D are X-ray computed tomography images of the wells of FIG.3A.

FIGS. 5A-5D are X-ray computed tomography images of the wells of FIG.2B.

FIG. 6A is a bright field microscopy image of the wells of FIG. 3A.

FIG. 6B is a bright field microscopy image of the wells of FIG. 2B.

FIG. 7 is a schematic cross-sectional view of an embodiment of areservoir including a plurality of wells.

FIG. 8 is a schematic side view of a deforming process for fabricationof thin wall wells.

DETAILED DESCRIPTION

Reference will now be made in greater detail to various embodiments ofthe subject matter of the present disclosure, some embodiments of whichare illustrated in the accompanying drawings. Like numbers used in thefigures refer to like components, steps and the like. However, it willbe understood that the use of a number to refer to a component in agiven figure is not intended to limit the component in another figurelabeled with the same number. In addition, the use of different numbersto refer to components is not intended to indicate that the differentnumbered components cannot be the same or similar to other numberedcomponents.

The present disclosure describes, among other things, cell cultureapparatuses having a structured bottom surface defining a shape of aplurality of wells or microwells. In some embodiments, a substrateforming the wells can comprise an exterior surface that defines anexternal surface of the apparatus. The shape of the external surface canbe controlled to facilitate imaging of cells within the wells inaccordance with various embodiments described herein.

In some embodiments, the wells may be configured such that cellscultured in the wells form spheroids. For example, the wells may benon-adherent to cells to cause the cells in the wells to associate witheach other and form spheres. The spheroids may expand to size limitsimposed by the geometry of the cells. In some embodiments, the wells maybe coated with an ultra-low binding material to make the wellsnon-adherent to cells.

In some embodiments, the inner surface of the wells may be non-adherentto cells. The wells may be formed from non-adherent material or may becoated with non-adherent material to form a non-adherent well. In someembodiments, the non-adherent material may be described as anultra-low-adhesion material. Examples of non-adherent material includeperfluorinated polymers, olefins, or like polymers or mixtures thereof.Other examples may include agarose, non-ionic hydrogels such aspolyacrylamides, or like materials or mixtures thereof. The combinationof, for example, non-adherent wells, well geometry, and gravity mayinduce cells cultured in the wells to self-assembly into spheroids.

However, well geometries that can be useful for culturing spheroids canbe difficult to image, either manually or via automated processes, withconventional microscopy techniques due to light distortions introducedthrough lens like effects by each individual well.

The well or well array design described herein may make image analysisof in vitro 3-dimensional spheroid based assays or spheroid productionpossible or more feasible. The cross-sectional profile of an individualwell may have an impact on quality of imaging capabilities, e.g.,microscope imaging capabilities. Specifically, controlling the wellthickness and outer shape of the well may help compensate for light pathdeviation during imaging to improve image quality and may make the cellculture system amenable to high content imaging screening. As a result,the well thickness and outer shape of the well may lead to a well thatis optically active due to the lens shape. In other words, the well maybe able to utilize one or a variety of light sources and still produceuniform illumination of the cells in the well. In some embodiments, animproved illumination may allow for a shorter focal length, which mayincrease the NA of the system and allow image acquisition at highermagnifications.

A variety of well characteristics may have a significant impact onimaging quality. For example, dimensions and shape of an interiorsurface of the well, dimensions and shape of an exterior surface of thewell, optical properties of the material defining the well, thethickness profile of the material defining the well, etc. can all play arole in high quality microscopy imaging. Additionally, the refractiveindex of a material may have a significant impact on imaging quality inboth reflective and transmittance microscopy applications. For example,in the case of many cell culture imaging applications, the most commonmaterial that is in contact with the interior surface of the well is awater-based solution with a refractive index of 1.33 and the most commonmaterial for the well fabrication is polystyrene with a refractive indexof 1.59. The differences in refractive indexes of the two materials maycause any incident light beam to deflect/reflect and may result in anegative impact on the microscope image quality.

One way to improve the quality of cell culture images may be to correctthe light distortion. The light distortion may be corrected bycontrolling and varying the well characteristics discussed above.Specifically, the dimensions and shape of the interior and exteriorsurfaces of the well and the thickness profile of the material definingthe well. Previously published fabrication methods have focused on thedimensions and shape of the interior surface of the well, especially,the interior surface that defines the dimensions of the 3D cellularaggregates. However, adjusting any of these characteristics in relationto one another may help to compensate for any light distortion that mayoccur during imaging (e.g., microscopy, etc.). More specifically, and asdescribed herein, the light distortion may be corrected by controllingthe shape and dimensions of the exterior surface of the well. In otherwords, the ability to change the shape and dimensions of the exteriorsurface may be utilized to help control the angle at which incidentlight exits the exterior surface.

A cell culture apparatus 100 including a plurality of wells 115 is shownin FIG. 1. The plurality of wells 115 may be defined by a substrate 110,e.g., a polymeric material. Each well 115 may define an interior surface120, an exterior surface 114, an upper aperture 118, a nadir 116, and anupper edge 121. The substrate 110 may define a thickness 111 between theinterior surface 120 and the exterior surface 114. The wells 115 mayhave a depth d defined by a height from the nadir 116 to the upperaperture 118. The wells 115 may also have a diametric dimension w, suchas a diameter, width, etc., across the well 115 defined by the upperaperture 118.

In some embodiments, the wells 115 described herein may define adiametric dimension w of about, e.g., greater than or equal to 100micrometers, greater than or equal to 300 micrometers, greater than orequal to 500 micrometers, greater than or equal to 800 micrometers,greater than or equal to 1200 micrometers, etc. or, less than or equalto 3000 micrometers, less than or equal to 2600 micrometers, less thanor equal to 2200 micrometers, less than or equal to 1800 micrometers,less than or equal to 1500 micrometers, etc., including ranges betweenany of the foregoing values. Such diametric dimensions can control thesize of a spheroid grown therein such that cells at the interior of thespheroid are maintained in a healthy state. In some embodiments, thewells 115 may define a depth d, by way of example, greater than or equalto 100 micrometers, greater than or equal to 300 micrometers, greaterthan or equal to 500 micrometers, greater than or equal to 800micrometers, greater than or equal to 1200 micrometers, etc. or, lessthan or equal to 3000 micrometers, less than or equal to 2600micrometers, less than or equal to 2200 micrometers, less than or equalto 1800 micrometers, less than or equal to 1500 micrometers, etc.,including ranges between any of the foregoing values. Of course, othersuitable dimensions may also be employed.

The exterior surface of the well may be a variety of shapes. Forexample, the shape of the exterior surface may be configured to correctfor refraction of light passing into the interior surface of the welland out of the exterior surface of the well or vice versa. In otherwords, the light passing out of the exterior surface of the well issubstantially parallel to the light passing into the interior surfaceand/or the shape of the exterior surface may be configured to minimizerefraction of light that passes between the interior and exteriorsurfaces and/or the exterior surface may be configured to transmit lightsubstantially parallel to a direction that the light was received by theinterior surface of the well. In some embodiments, the well contains acell culture medium, and the shape of the exterior surface corrects forrefraction.

The thickness of the substrate between the interior surface of the welland the exterior surface of the well may vary. For example, thethickness of the substrate may be configured to correct for refractionof light passing into the interior surface of the well and out of theexterior surface of the well or vice versa. In other words, the lightpassing out of the exterior surface of the well is substantiallyparallel to the light passing into the interior surface or the thicknessof the substrate may be configured to minimize refraction of light thatpasses there between. In some embodiments, the well contains a cellculture medium when the thickness of the substrate corrects forrefraction.

The cross-sections of two wells 200 that define an exterior surface 214that does not correct for refraction are shown in FIGS. 2A and 2B. Inother words, light that enters 201 the interior surface 220 of the well200 is not parallel with light that exits 202 the exterior surface 214of the well 200.

As shown in FIG. 2A, the thickness of the substrate 210 proximate thenadir 216 is less than the thickness of the substrate 210 proximate theupper edge 221 of the well 200. The thickness of the substrate 210proximate the upper edge 221 of the well 200 may be defined as athickness between the interior surface 220 and the exterior surface 214on a same plane as the upper aperture 218. As shown in FIG. 2B, theexterior surface 214 of the well 200 defines a rectangular shaped bottomof the substrate 210 that creates a flat exterior surface of the well200.

In some embodiments, the thickness and shape of the substrate, e.g., apolymeric material, that defines the well may be configured to correctfor refraction of light passing into the interior surface of the welland out of the exterior surface of the well. The cross-sections of twoembodiments of wells 115 that define an exterior surface 114 that doescorrect for refraction are shown in FIGS. 3A and 3B. In other words,light that enters 201 the interior surface 120 of the well 115 isparallel with light that exits 202 the exterior surface 114 of the well115. In yet other words, a shape of the interior surface 120 of the well115 and a shape of the exterior surface 114 of the well 115 areconfigured to minimize the effects of the refraction of light thatpasses there between.

As shown in FIGS. 3A and 3B, the thickness 111 of the substrate 110proximate the nadir 116 may be greater than or equal to the thickness109 of the substrate 110 proximate the upper aperture 118. The thicknessof the substrate 110 proximate the nadir 116 may be defined as adistance between the interior surface 120 and the exterior surface 114at a lowest point of the well 115. The thickness of the substrate 110proximate the upper aperture 118 may be defined as a thickness betweenthe interior surface 120 and the exterior surface 114 on a same plane asthe upper aperture 118.

Specifically, as shown in FIG. 3A, the thickness of the substrate 110remains constant from proximate the upper aperture 118 to the nadir 116and, as shown in FIG. 3B, the thickness 111 of the substrate 110proximate the nadir 116 is greater than the thickness 109 of thesubstrate 110 proximate the upper aperture 118. Also, as shown in FIG.3A, the thickness 111 of the substrate 110 proximate to the nadir 116may be equal to the thickness 109 of the substrate 110 proximate theupper aperture 118. The substrate thicknesses shown in FIGS. 3A and 3Ballow for an incoming light 201 entering the interior surface 120 to besubstantially parallel to an outgoing light 202 leaving the exteriorsurface 114.

In other embodiments, the substrate thickness may be described asincreasing continuously from proximate the upper aperture to the nadir(e.g., FIG. 3B). The thickness of the substrate proximate any locationfrom the upper aperture to the nadir may be defined by a thickness of,e.g., greater than or equal to 5 micrometers, greater than or equal to10 micrometers, greater than or equal to 20 micrometers, greater than orequal to 40 micrometers, greater than or equal to 60 micrometers, etc.or, less than or equal to 100 micrometers, less than or equal to 90micrometers, less than or equal to 80 micrometers, less than or equal to65 micrometers, less than or equal to 50 micrometers, etc., includingranges between any of the foregoing values. In some embodiments, thethickness is about 1000 micrometers or less. In some embodiments, thethickness is in a range from 10 micrometers to 1000 micrometers.

In some embodiments, the well may define an axis 105 between the nadirand a center of the upper aperture and the well may be rotationallysymmetrical about the axis 105 (see, e.g., FIG. 1). For example, ahemispherical shape may define the well. The hemispherical shape may bedefined by a radius of about, e.g., greater than or equal to 50micrometers, greater than or equal to 150 micrometers, greater than orequal to 250 micrometers, greater than or equal to 400 micrometers,greater than or equal to 600 micrometers, etc. or, less than or equal to1500 micrometers, less than or equal to 1300 micrometers, less than orequal to 1100 micrometers, less than or equal to 900 micrometers, lessthan or equal to 750 micrometers, etc.

Orthogonal views of 3D datasets of X-ray computed tomography images ofwells of generally as depicted in FIG. 3A are shown in FIGS. 4A-4D. Theimages depict wells 115 defining a convex exterior surface 114 asdescribed in FIG. 3A. FIG. 4A depicts a cross sectional view of threecomplete wells 115 along horizontal line 117, shown in FIG. 4C. FIG. 4Cis a top view of a portion of a cell culture apparatus with an array ofwells 115. FIG. 4B is a cross sectional view of wells 115 along verticalline 119 shown in FIG. 4C. FIG. 4D is a reconstituted 3D image of aportion of a cell culture apparatus with an array of wells 115.

Orthogonal views of 3D datasets of X-ray computed tomography images ofwells of generally as depicted in FIG. 2B are shown in FIGS. 5A-5D. Theimages depict wells 115 defining a flat exterior surface 114 asdescribed in FIG. 2B. FIG. 5A depicts a cross sectional view of wells115 along horizontal line 117 shown in FIG. 5C. FIG. 5C of a portion ofa cell culture apparatus with an array of wells 115. FIG. 5B is a crosssectional view of wells 115 along vertical line 119 shown in FIG. 5C.FIG. 5D is a reconstituted 3D image of a portion of a cell cultureapparatus with an array of wells 115.

Bright field microscopy images of wells 115 having shapes generally inaccordance with FIGS. 3A and 2B are shown in FIGS. 6A and 6B,respectively. The microscopy images of FIG. 6A shows light that passedthrough wells having a shape as depicted in FIG. 3A. The microscopyimages of FIG. 6B shows light that passed through wells having a shapeas depicted in FIG. 2B. The shape of the well as depicted in FIG. 3A didnot substantially reflect/deflect and thus yielded a relatively uniformsignal across all wells as compared to the signal across wells having ashape as depicted in FIG. 2B. More light was received by the microscopecamera for wells having a shape as depicted in FIG. 3A (see FIG. 6A)than the wells having a shape as depicted in FIG. 2B as shown inmicroscopy images of FIG. 6B. In other words, the well microscopy imagesof FIG. 6B depicts that more light was scattered, as shown by the darkrings, than the well microscopy images of FIG. 6A.

As shown in FIG. 7, the cell culture apparatus 700 may include areservoir 725. The reservoir may include a bottom 705 and an enclosingsidewall 720. The bottom 705 may be defined by a plurality of wells 715.Each well 715 may have similar characteristics as wells described herein(see, e.g., FIGS. 1, 3A, and 3B).

In some embodiments, the exterior surface of the well is optimizedthrough ray tracing for diffraction limited imaging performance whenviewed under high resolution microscopy (e.g., bright field,fluorescence, confocal, or other microscopy modalities). For example,with reference to FIG. 2A and FIG. 2B, the exterior surface 214 isoptimized through ray tracing.

To illustrate this approach, an interior surface of a polystyrene wellmay be a hemisphere with a radius of 500 micrometers and a centerthickness of 150 micrometers. The diameter of a spheroid may be 300micrometers, and a 20× microscope with an objective numerical apertureof 0.4 is employed. There may be a number of image points acrosspositions of the spheroid, for example, center, 50 micrometers from thecenter, 100 micrometers from the center, and 150 micrometers from thecenter. In such instances, most images taken will be sub-optimum. Spotdiagrams can be generated from the different field positions andcompared to the diffraction limited Airy circle at the image plane toassess image quality. If the exterior surface is flat, as illustrated inFIG. 2B, the spot diameters across the field are a few times larger thanthe diffraction limit, indicating poor image quality. When the well hasa uniform thickness, the image quality is considerably better than theprevious case. However, diffraction limited imaging performance isbarely achieved within the center 50 micrometer radius. Outside thisfield of view, astigmatism deteriorates the image quality. However, byoptimizing the radius of curvature of the exterior surface to 0.518 mm,the image quality can achieve diffraction limited performance across theentire spheroid diameter, although a small amount of distortion andastigmatism still exist. To further optimize the image quality, anaspheric exterior surface is used. With a radius of curvature R=0.682 mmand a conic constant of K=−3.09, the residual aberration and distortionthroughout the entire field of interest are removed. The conic surfaceis given by:

y ²−2Rx+(K+1)x ²=0.

Diffraction limited performance is also maintained in the entire volumeof the spheroid. This enables high resolution confocal imaging in anylocations within the spheroid. The actual magnification is 21.5× due tothe refractive effect of the surface.

In some embodiments, nested wells are employed, whereby a first well orlayer of wells is present above a second well or layer of wells. Wellsidewalls of each well are selected such that light passing through twoor more layers of wells remains substantially parallel to the originallight.

Any suitable process can be used to fabricate cell culture apparatuseshaving wells as described herein. For example, a substrate can be moldedto form the well or structured surface, a substrate film can be embossedto form the well or structured surface, or the like. In someembodiments, a deforming process is used to fabricate wells as describedherein.

For example and with reference to FIG. 8, a schematic side view of adeforming process for fabrication of wells is shown. For example, FIG. 8illustrates a hot embossing and film deforming process 800 for thefabrication of thin wall wells. The process uses a thin film 820 andapplies heat and pressure 810 down onto the thin film 820 into the mold830. The thin film 820 may have a specific thickness that results in agiven thickness attributed to different sections of the wells. Forexample, a 70 micrometer thin film after going through a process of hotembossing and film deforming may have a uniform thickness of 25micrometers at a bottom part of the well and upper part of the well.This outcome is similar to the well shown in FIG. 3A, which cansufficiently correct for light refraction. As a result, the hotembossing and film deforming process may be actively controlled duringwell fabrication to form wells that correct light refractionsufficiently similar to those in FIGS. 3A-3B. The well fabricationprocess may also be performed in planar configuration or as a roll toroll process.

Cell culture apparatuses having wells or structured surfaces asdescribed herein can be formed from any suitable material. Preferably,materials intended to contact cells or culture media are compatible withthe cells and the media. Typically, cell culture components (e.g.,wells) are formed from polymeric material. Examples of suitablepolymeric materials include polystyrene, polymethylmethacrylate,polyvinyl chloride, polycarbonate, polysulfone, polystyrene copolymers,fluoropolymers, polyesters, polyamides, polystyrene butadienecopolymers, fully hydrogenated styrenic polymers, polycarbonate PDMScopolymers, and polyolefins such as polyethylene, polypropylene,polymethyl pentene, polypropylene copolymers and cyclic olefincopolymers, and the like.

Cells cultured in three dimensions, such as spheroids, can exhibit morein vivo like functionality than their counterparts cultured in twodimensions as monolayers. In two dimensional cell culture systems, cellscan attach to a substrate on which they are cultured. However, whencells are grown in three dimensions, such as spheroids, the cellsinteract with each other rather than attaching to the substrate. Cellscultured in three dimensions more closely resemble in vivo tissue interms of cellular communication and the development of extracellularmatrices. Spheroids thus provide a superior model for cell migration,differentiation, survival, and growth and therefore provide bettersystems for research, diagnostics, and drug efficacy, pharmacology, andtoxicity testing.

In some embodiments, the devices are configured such that cells culturedin the devices form spheroids. For example, the wells in which cells aregrown can be non-adherent to cells to cause the cells in the wells toassociate with each other and form spheres. The spheroids expand to sizelimits imposed by the geometry of the wells. In some embodiments, thewells are coated with an ultra-low binding material to make the wellsnon-adherent to cells.

Examples of non-adherent material include perfluorinated polymers,olefins, or like polymers or mixtures thereof. Other examples includeagarose, non-ionic hydrogels such as polyacrylamides, polyethers such aspolyethylene oxide and polyols such as polyvinyl alcohol, or likematerials or mixtures thereof. The combination of, for example,non-adherent wells, well geometry (e.g., size and shape), and/or gravityinduce cells cultured in the wells to self-assemble into spheroids. Somespheroids maintain differentiated cell function indicative of a more invivo-like, response relative to cells grown in a monolayer. Other cellstypes, such as mesenchymal stromal cells, when cultured as spheroidsretain their pluripotency.

In some embodiments, the systems, devices, and methods herein compriseone or more cells. In some embodiments, the cells are cryopreserved. Insome embodiments, the cells are in three dimensional culture. In somesuch embodiments, the systems, devices, and methods comprise one or morespheroids. In some embodiments, one or more of the cells are activelydividing. In some embodiments, the systems, devices, and methodscomprise culture media (e.g., comprising nutrients (e.g., proteins,peptides, amino acids), energy (e.g., carbohydrates), essential metalsand minerals (e.g., calcium, magnesium, iron, phosphates, sulphates),buffering agents (e.g., phosphates, acetates), indicators for pH change(e.g., phenol red, bromo-cresol purple), selective agents (e.g.,chemicals, antimicrobial agents), etc.). In some embodiments, one ormore test compounds (e.g., drug) are included in the systems, devices,and methods.

A wide variety of cell types may be cultured. In some embodiments, aspheroid contains a single cell type. In some embodiments, a spheroidcontains more than one cell type. In some embodiments, where more thanone spheroid is grown, each spheroid is of the same type, while in otherembodiments, two or more different types of spheroids are grown. Cellsgrown in spheroids may be natural cells or altered cells (e.g., cellcomprising one or more non-natural genetic alterations). In someembodiments, the cell is a somatic cell. In some embodiments, the cellis a stem cell or progenitor cell (e.g., embryonic stem cell, inducedpluripotent stem cell) in any desired state of differentiation (e.g.,pluripotent, multi-potent, fate determined, immortalized, etc.). In someembodiments, the cell is a disease cell or disease model cell. Forexample, in some embodiments, the spheroid comprises one or more typesof cancer cells or cells that can be induced into a hyper-proliferativestate (e.g., transformed cells). Cells may be from or derived from anydesired tissue or organ type, including but not limited to, adrenal,bladder, blood vessel, bone, bone marrow, brain, cartilage, cervical,corneal, endometrial, esophageal, gastrointestinal, immune system (e.g.,T lymphocytes, B lymphocytes, leukocytes, macrophages, and dendriticcells), liver, lung, lymphatic, muscle (e.g., cardiac muscle), neural,ovarian, pancreatic (e.g., islet cells), pituitary, prostate, renal,salivary, skin, tendon, testicular, and thyroid. In some embodiments,the cells are mammalian cells (e.g., human, mice, rat, rabbit, dog, cat,cow, pig, chicken, goat, horse, etc.).

The cultured cells find use in a wide variety of research, diagnostic,drug screening and testing, therapeutic, and industrial applications.

In some embodiments, the cells are used for production of proteins orviruses. Systems, devices, and methods that culture large numbers ofspheroids in parallel are particularly effective for protein production.Three-dimensional culture allows for increased cell density, and higherprotein yield per square centimeter of cell growth surface area. Anydesired protein or viruses for vaccine production may be grown in thecells and isolated or purified for use as desired. In some embodiments,the protein is a native protein to the cells. In some embodiments, theprotein is non-native. In some embodiments, the protein is expressedrecombinantly. Preferably, the protein is overexpressed using anon-native promoter. The protein may be expressed as a fusion protein.In some embodiments, a purification or detection tag is expressed as afusion partner to a protein of interest to facilitate its purificationand/or detection. In some embodiments, fusions are expressed with acleavable linker to allow separation of the fusion partners afterpurification.

In some embodiments, the protein is a therapeutic protein. Such proteinsinclude, but are not limited to, proteins and peptides that replace aprotein that is deficient or abnormal (e.g., insulin), augment anexisting pathway (e.g., inhibitors or agonists), provide a novelfunction or activity, interfere with a molecule or organism, or deliverother compounds or proteins (e.g., radionuclides, cytotoxic drugs,effector proteins, etc.). In some embodiments, the protein is animmunoglobulin such as an antibody (e.g., monoclonal antibody) of anytype (e.g., humanized, bi-specific, multi-specific, etc.). Therapeuticprotein categories include, but are not limited to, antibody-baseddrugs, Fc fusion proteins, anticoagulants, antigens, blood factor, bonemorphogenetic proteins, engineered protein scaffolds, enzymes, growthfactors, hormones, interferons, interleukins, and thrombolytics.Therapeutic proteins may be used to prevent or treat cancers, immunedisorders, metabolic disorders, inherited genetic disorders, infections,and other diseases and conditions.

In some embodiments, the protein is a diagnostic protein. Diagnosticproteins include, but are not limited to, antibodies, affinity bindingpartners (e.g., receptor-binding ligands), inhibitors, antagonists, andthe like. In some embodiments, the diagnostic protein is expressed withor is a detectable moiety (e.g., fluorescent moiety, luminescent moiety(e.g., luciferase), colorimetric moiety, etc.).

In some embodiments, the protein is an industrial protein. Industrialproteins include, but are not limited to, food components, industrialenzymes, agricultural proteins, analytical enzymes, etc.

In some embodiments, the cells are used for drug discovery,characterization, efficacy testing, and toxicity testing. Such testingincludes, but is not limited to, pharmacological effect assessment,carcinogenicity assessment, medical imaging agent characteristicassessment, half-life assessment, radiation safety assessment,genotoxicity testing, immunotoxicity testing, reproductive anddevelopmental testing, drug interaction assessment, dose assessment,adsorption assessment, disposition assessment, metabolism assessment,elimination studies, etc. Specific cells types may be employed forspecific tests (e.g., hepatocytes for liver toxicity, renal proximaltubule epithelial cells for nephrotoxicity, vascular endothelial cellsfor vascular toxicity, neuronal and glial cells for neurotoxicity,cardiomyocytes for cardiotoxicity, skeletal myocytes for rhabdomyolysis,etc.). Treated cells may be assessed for any number of desiredparameters including, but not limited to, membrane integrity, cellularmetabolite content, mitochondrial functions, lysosomal functions,apoptosis, genetic alterations, gene expression differences, and thelike.

In some embodiments, the cell culture devices are a component of alarger system. In some embodiments, the system comprises a plurality(e.g., 2, 3, 4, 5, . . . , 10, . . . , 20, . . . , 50, . . . , 100, . .. , 1000, etc.) of such cell culture devices. In some embodiments, thesystem comprises an incubator for maintaining the culture devices atoptimal culture conditions (e.g., temperature, atmosphere, humidity,etc.). In some embodiments, the system comprises detectors for imagingor otherwise analyzing cells. Such detectors include, but are notlimited to, fluorimeters, luminometers, cameras, microscopes, platereaders (e.g., PERKIN ELMER ENVISION plate reader; PERKIN ELMER VIEWLUXplate reader), cell analyzers (e.g., GE IN Cell Analyzer 2000 and 2200;THERMO/CELLOMICS CELLNSIGHT High Content Screening Platform), andconfocal imaging systems (e.g., PERKIN ELMER OPERAPHENIX high throughputcontent screening system; GE INCELL 6000 Cell Imaging System). In someembodiments, the system comprises perfusion systems or other componentsfor supplying, re-supplying, and circulating culture media or othercomponents to cultured cells. In some embodiments, the system comprisesrobotic components (e.g., pipettes, arms, plate movers, etc.) forautomating the handing, use, and/or analysis of culture devices.

All scientific and technical terms used herein have meanings commonlyused in the art unless otherwise specified. The definitions providedherein are to facilitate understanding of certain terms used frequentlyherein and are not meant to limit the scope of the present disclosure.

As used herein, singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a “structured bottom surface” includes exampleshaving two or more such “structured bottom surfaces” unless the contextclearly indicates otherwise.

As used in this specification and the appended claims, the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise. The term “and/or” means one or all of thelisted elements or a combination of any two or more of the listedelements.

As used herein, “have”, “has”, “having”, “include”, “includes”,“including”, “comprise”, “comprises”, “comprising” or the like are usedin their open ended inclusive sense, and generally mean “include, butnot limited to”, “includes, but not limited to”, or “including, but notlimited to”.

“Optional” or “optionally” means that the subsequently described event,circumstance, or component, can or cannot occur, and that thedescription includes instances where the event, circumstance, orcomponent, occurs and instances where it does not.

The words “preferred” and “preferably” refer to embodiments of thedisclosure that may afford certain benefits, under certaincircumstances. However, other embodiments may also be preferred, underthe same or other circumstances. Furthermore, the recitation of one ormore preferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the inventive technology.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, examples include from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.). Where a range of values is “greater than”,“less than”, etc. a particular value, that value is included within therange.

Any direction referred to herein, such as “top,” “bottom,” “left,”“right,” “upper,” “lower,” “above,” below,” and other directions andorientations are described herein for clarity in reference to thefigures and are not to be limiting of an actual device or system or useof the device or system. Many of the devices, articles or systemsdescribed herein may be used in a number of directions and orientations.Directional descriptors used herein with regard to cell cultureapparatuses often refer to directions when the apparatus is oriented forpurposes of culturing cells in the apparatus.

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatany particular order be inferred. Any recited single or multiple featureor aspect in any one claim can be combined or permuted with any otherrecited feature or aspect in any other claim or claims.

It is also noted that recitations herein refer to a component being“configured” or “adapted to” function in a particular way. In thisrespect, such a component is “configured” or “adapted to” embody aparticular property, or function in a particular manner, where suchrecitations are structural recitations as opposed to recitations ofintended use. More specifically, the references herein to the manner inwhich a component is “configured” or “adapted to” denotes an existingphysical condition of the component and, as such, is to be taken as adefinite recitation of the structural characteristics of the component.

While various features, elements or steps of particular embodiments maybe disclosed using the transitional phrase “comprising,” it is to beunderstood that alternative embodiments, including those that may bedescribed using the transitional phrases “consisting” or “consistingessentially of,” are implied. Thus, for example, implied alternativeembodiments to a cell culture apparatus comprising a structured bottomsurface, one or more sidewalls, a top and a port include embodimentswhere a cell culture apparatus consists of a structured bottom surface,one or more sidewalls, a top and a port and embodiments where a cellculture apparatus consists essentially of a structured bottom surface,one or more sidewalls, a top and a port.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the present inventivetechnology without departing from the spirit and scope of thedisclosure. Since modifications, combinations, sub-combinations andvariations of the disclosed embodiments incorporating the spirit andsubstance of the inventive technology may occur to persons skilled inthe art, the inventive technology should be construed to includeeverything within the scope of the appended claims and theirequivalents.

What is claimed is:
 1. A cell culture apparatus comprising: a substratedefining a well, wherein the well defines an interior surface having ahemispheric shape, an exterior surface, an upper aperture and a nadir;wherein the substrate defines a thickness between the interior surfaceand the exterior surface; wherein the thickness increases continuouslyfrom proximate the upper aperture to the nadir.
 2. The cell cultureapparatus of claim 1, wherein the thickness of the substrate proximateto the nadir is greater than the thickness of the substrate proximate tothe upper aperture.
 3. The cell culture apparatus of claim 1, whereinthe well defines an axis between the nadir and a center of the upperaperture, wherein the well is rotationally symmetrical about the axis.4. The cell culture apparatus according to claim 1, wherein the upperaperture defines a distances across the upper aperture, wherein thedistance across the upper aperture is in a range from 100 micrometers to3000 micrometers.
 5. The cell culture apparatus according to claim 1,wherein the thickness of the substrate at any location from proximatethe upper aperture to the nadir is in a range from 10 micrometers to1000 micrometers.
 6. The cell culture apparatus of claim 1, whereininterior surface is defined by a hemispherical shape, wherein thehemispherical shape defines a radius in a range from 50 micrometers to1500 micrometers.
 7. The cell culture apparatus according to claim 1,wherein the substrate comprises polystyrene.
 8. The cell cultureapparatus according claim 1, wherein exterior surface is configured totransmit light substantially parallel to a direction that the light wasreceived by the interior surface when the well contains a cell culturemedium.
 9. The cell culture apparatus according to claim 1, wherein ashape of the interior surface and a shape of the exterior surface areconfigured to minimize refraction of light that passes there betweenwhen the well contains a cell culture medium.
 10. The cell cultureapparatus according to claim 1, wherein the well is non-adherent tocells.
 11. The cell culture apparatus according to claim 1, wherein theinterior surface is configured such that cells cultured therein form aspheroid.
 12. A cell culture apparatus comprising: a reservoircomprising a bottom and an enclosing sidewall, wherein the bottom isdefined by a plurality of wells, wherein each well of the plurality ofwells defines an interior surface, an exterior surface, an upperaperture and a nadir, wherein each well defines a thickness between theinterior surface and the exterior surface, wherein the thicknessincreases continuously from proximate the upper aperture to the nadir.13. The cell culture apparatus of claim 12 wherein the thickness isconfigured to correct for refraction of light passing into the interiorsurface and out of the exterior surface when the well contains awater-based composition.
 14. The cell culture apparatus of claim 12wherein a shape of the exterior surface is configured to correct forrefraction of light passing into the interior surface and out of theexterior surface.
 15. The apparatus of claim 1, wherein said exteriorsurface has an aspheric exterior surface.
 16. Use of the apparatus ofclaim 1 for the growth of a spheroid.
 17. Use of the apparatus of claim12 for the imaging of a cell in said well.
 18. The use of claim 17,wherein said cell is in a spheroid.